Growth conditions influence at structural and virulence characteristics of Candida and Streptococcus biofilms developed on in vitro models of human oral mucosa

Abstract

The opportunistic pathogen Candida albicans and streptococci of the Mitis group form complex communities in multiple oral sites, where the environment and nutrient availability change constantly. We aimed to study structural and virulence characteristics of Candida albicans biofilms in the presence or absence of S. oralis, growing on a three-dimensional model of human oral mucosa, under different conditions: (1) moisture of mucosal surface (wet or semi dry), (2) nutrient availability (BHI supplementation on culture media) and (3) hyphal morphotype (hyphae or pseudohyphae). For this it was used a three-dimensional model of the human oral mucosa formed by immortalized oral keratinocytes (OKF6-TERT2 or SCC15 cell lines) on a fibroblast-embedded collagenous matrix to grow biofilms. Infections were carried out using Streptococcus oralis 34, a C. albicans reference strain and pseudohyphal mutants with a homozygous deletion of the ndt80, or tup1 gene. Determination of biofilm biovolume and structure was done by confocal scanning laser microscopy with biofilms stained by immunofluorescence using an anti-Candida antibody and a Streptococcus probe. As determinant of virulence, 5-¼m-thick tissue sections were stained same way or with hematoxylin and eosin in order to detect invasion of microorganisms. Also tissue damage was measured by lactate dehydrogenase release in the culture media. Statistical analyses were performed using SigmaPlot 12 software at 5% significance level. Data were evaluated by analysis of variance (ANOVA) and when statistical significances were found, all pairwise multiple comparison procedures were performed with Bonferroni t-test, with ± = 5%. Under wet conditions C. albicans extended long intertwined hyphae, forming a homogeneous surface biofilm. Mixed biofilms had a stratified structure, with S. oralis growing in close contact to the mucosa and C. albicans growing on the bacterial surface. Under semi-dry conditions, C. albicans formed localized foci of dense growth from which hyphae extended radially to intertwine with hyphae from adjacent foci. In mixed biofilms this promoted focal growth of S. oralis co-localizing with C. albicans. Although Candida biofilm biovolume was significantly greater under wet conditions (P<0.001), there was minimal tissue invasion compared to semidry conditions where the epithelial barrier was completely destroyed. Supplementing the infection medium with nutrients under semidry conditions did not change the biofilm architecture but intensified focal growth and increased biofilm biovolume and tissue invasion/damage (P<0.001), proportionally to the tested concentrations. Pseudohyphal mutants formed defective mixed biofilms, with most S. oralis in contact with the epithelial surface, below the pseudohyphal mass. Interestingly, the presence of S. oralis promoted fungal invasion and tissue damage under all conditions. Moisture, nutrient availability, hyphal morphotype and presence of S. oralis strongly affect architecture and virulence of mucosal fungal biofilms.